The present invention relates to a method and apparatus for analysing an image.
A number of industrial applications require the analysis of an image of a localised feature. In particular, it may be desirable to analyze the image to determine the average brightness of the localised feature. However, the image of the localised feature may be more spread out than the original feature itself (for instance due to errors in the imaging process such as optical flare, or light from the original feature being reflected over adjacent areas). This causes the problem that the average brightness is calculated over an area greater than the actual area of the original localised feature, and as a result the calculated average brightness is erroneously low.
This problem has conventionally been addressed by reducing the number of pixels which are averaged. For instance, only pixels lying above a fixed lower threshold pixel value may be averaged. The disadvantage with this approach is that if the image of the localised area has low brightness, pixels which should be averaged may fall below the lower threshold pixel value. This results in a reduction in dynamic range and deviation from linearity in the average brightness measurement.
According to a first aspect of the present invention, there is provided a method of analysing an image to obtain an image value, the image comprising a defined array of pixel values, the method comprising
(1) determining the highest pixel value in the image;
(2) calculating a lower threshold pixel value from the highest pixel value determined in step (1) in accordance with a predetermined algorithm; and
(3) obtaining the image value by statistically analysing the pixel values in the image which lie in a range defined by the lower threshold pixel value calculated in step (2).
The invention solves the problem outlined above by calculating the lower threshold pixel value from the highest pixel value. In contrast to the conventional approach which uses a fixed lower threshold pixel value, the invention uses a variable lower threshold pixel value which is related to the highest pixel value in the image. This results in increased linearity and increased dynamic range.
Any suitable statistical method may be employed in step (3), but preferably the image value is obtained in step (3) by averaging the pixel values. The average may be a median or mode average but preferably the average is a mean average. Alternatively, the statistical analysis may involve either summing or integrating the pixel values which lie within the defined range.
Typically, the pixel values are related to the intensity of radiation from an original image, and in a preferred embodiment the method further comprises detecting radiation from the original image to generate the array of pixel values. The total radiation intensity may be detected, or alternatively the intensity in one or more selected wavelength ranges may be detected.
A further problem can occur if the image contains erroneously high pixel values (eg. bright spots). This can result in the calculated image value being too high. This problem can be dealt with by calculating an upper pixel threshold value, and/or by removing high pixel values before the algorithm is performed, as discussed below.
Therefore, the method may further comprise calculating an upper pixel threshold value from the highest pixel value determined in step (1) in accordance with a second predetermined algorithm, wherein step (3) comprises statistically analysing the pixel values in the image which lie in a range defined by the upper and lower pixel threshold values. In some cases, the bright spots may be an order of magnitude higher than the image values in the rest of the image. Where the image is derived from an assay reaction site, this may be caused by cosmic rays. This will result in an erroneously large highest pixel value being used. Therefore, in this case, the method preferably comprises previously defining the defined array of pixel values by reviewing an original array of pixel values, and removing one or more of the highest pixel values in the original array of pixel values. This may be done automatically by statistical analysis, or by manual inspection. Both methods remove the bright spots from the averaging process and consequently improve accuracy.
The predetermined algorithm(s) may calculate the threshold(s) as any fixed function of the highest pixel value. For instance the thresholds may be calculated as the square root of the highest pixel value. Alternatively the highest pixel value may be input to a look-up table which has been previously loaded with a range of threshold values. However, preferably the lower and/or upper threshold pixel values are calculated as a predetermined percentage of the highest pixel value determined in step (1).
Typically, the lower threshold pixel value is between 50% and 90% of the highest pixel value determined in step (1), preferably substantially 80%.
Typically, the upper threshold pixel value is between 97% and 99% of the highest pixel value determined in step (1), preferably substantially 98%.
Background noise may be present in the pixel values resulting in a reduction in accuracy and dynamic range. Preferably, the background is removed from the pixel values, for instance by a method of mathematical morphology such as erosion, dilation, opening and/or thresholding. The background may be removed before or after step (1) or step (3).
In accordance with a second aspect of the present invention, there is provided a method of analysing an image to obtain a plurality of image values, the image comprising an array of pixel values, the method comprising dividing the image into a plurality of regions, and obtaining an image value from each region by a method according to the first aspect of the invention.
The second aspect of the present invention enables an image of a plurality of localised features to be analyzed.
Typically, the image is an image of a localised feature. For instance the localised feature may comprise a feature in a satellite image of the earth""s surface, or a feature in a telescope image. However preferably the localised feature comprises a reaction site containing a reactive species which reacts with an analyte and which has been exposed to a test sample.
In accordance with a third aspect of the present invention, there is provided a method of analysing a localised reaction site containing a reactive species which reacts with an analyte and which has been exposed to a test sample, the method comprising detecting radiation from the reaction site to generate an image comprising an array of pixel values, the radiation being indicative of the presence or absence of the analyte in the test sample; and analysing the image by a method according to the first aspect of the invention, thereby obtaining an image value indicative of the presence or absence of the analyte in the test sample.
The third aspect of the present invention provides improved dynamic range and linearity in the analysis of an assay reaction site. Typically, the image value is an experimental parameter such as a relative light unit (RLU) value.
In accordance with a fourth aspect of the present invention, there is provided a method of analysing a plurality of localised reaction sites, each containing a reactive species which reacts with a respective analyte and each having been exposed to a test sample, the method comprising detecting radiation from the reaction sites to generate an image comprising an array of pixel values, the radiation from each reaction site being indicative of the presence or absence of a respective analyte in the test sample; dividing the image into a plurality of image regions each corresponding with a respective one of the reaction sites; and analysing each image region by a method according to the first aspect of the invention, thereby obtaining a plurality of image values each being indicative of the presence or absence of a respective analyte in the test sample.
The fourth aspect of the invention enables a multi-analyte assay to be processed quickly and efficiently.
Typically, the or each reaction site is located on a solid state substrate, and the or each reactive species comprises a ligand covalently bonded to the substrate.
Typically, the surface of the substrate between the reaction sites is inert with respect to analyte.
The image may be generated by illuminating the or each reaction site and detecting reflected or transmitted radiation. However, preferably the radiation comprises chemiluminescent, bioluminescent or fluorescent radiation emitted from the or each reaction site. In this case, a filter may be employed so as to only detect the narrow wavelength band of interest.
In accordance with a fifth aspect of the present invention, there is provided a method of performing an assay, the method comprising exposing one or more localised reaction sites to a test sample, each reaction site containing a reactive species which reacts with a respective analyte; and analysing the or each reaction site by a method according to the third or fourth aspects of the present invention.
According to a sixth aspect of the present invention, there is provided apparatus for analysing an image to obtain an image value, the image comprising a defined array of pixel values, the apparatus comprising
(1) means for determining the highest pixel value in the image;
(2) means for calculating a lower threshold pixel value from the highest pixel value in accordance with a predetermined algorithm; and
(3) means for obtaining the image value by statistically analysing the pixel values in the image which lie in a range defined by the lower threshold pixel value.
According to a seventh aspect of the present invention, there is provided apparatus for analysing one or more reaction sites each containing a reactive species which reacts with an analyte and which has been exposed to a test sample, the apparatus comprising means for detecting radiation from the or each reaction site to generate an image comprising an array of pixel values; and means for analysing the image by a method according to the first or second aspect of the present invention, thereby obtaining one or more image values each being indicative of the presence or absence of a respective analyte in the test sample.
According to an eighth aspect of the present invention, there is provided apparatus for performing an assay, the apparatus comprising means for exposing one or more localised reaction sites to a test sample, each reaction site containing a reactive species which reacts with a respective analyte; and apparatus according to the seventh aspect of the present invention for analysing the or each reaction site.